Browsing by Subject "G protein coupled receptor"

β-Adrenergic receptors (βAR) are one of the key modulators of cardio-pulmonary functions and belong to a large family of membrane proteins, termed as G-protein coupled receptors (GPCRs). β-blockers (βAR antagonists) and βAR agonists are the mainstay treatments for heart failure and asthma respectively, which reflects the significance of βARs as therapeutic targets. The binding of catecholamines (e.g. adrenaline) to βARs activates intracellular transducer proteins such as hetero trimeric GTP binding proteins (G-proteins) or β-arrestins (βarr), which results in the regulation of cardiac output and bronchodilation.

The bifurcated signaling pathways initiated by G-protein and β-arrestin downstream of βAR, as well as other members in the GPCR family can be selectively activated, a phenomenon termed as ‘biased agonism’. Biased ligands, which can pharmacologically separate these pathways, are of major therapeutic interest due to their potential for improving the specificity of drug actions. For βAR, biased agonism towards β-arrestin is expected to render cardo-protective benefits, while selective activation of G proteins is hypothesized to subdue major side effects from current asthma therapy. Therefore, elucidation of how βARs can preferentially interact with their transducers is at the core of developing the next generation therapeutics, beyond conventional β-blockers and agonists.

Thus far, the exact mechanism behind GPCR biased agonism remains obscure. The leading hypothesis in the field is that GPCRs adopt distinct conformations that preferentially couple to G proteins or β-arrestins. In order to test this hypothesis, we developed and established a G protein biased mutant β2AR (Chapter 2), since efficacious biased ligands for this receptor are yet to be found. Subsequent assessment of GPCR kinase (GRK)-mediated phosphorylation states of this mutant receptor and phosphorylation rescue experiments revealed unexpected findings that contradict the initial hypothesis (Chapter 3). Next, we initiated a biophysical characterization of this mutant β2AR (Chapter 4) to comprehend the conformational and structural basis for its apparent biased phenotype. The cumulative insight gained from experiments described in chapters 2-4 highlight the underappreciated role of GRKs in determining GPCR biased agonism – the mutant β2AR is biased towards G protein due to conformational selection against GRKs, rather than β-arrestins. Furthermore, to obtain a comprehensive understanding of biased agonism, we devised a strategy to map the interface between β2AR-β-arrestin, which can also be used to form stable complexes for further biophysical characterizations (Chapter 5). In summary, this dissertation improves the current understanding of the molecular mechanism behind biased agonism at the prototypical GPCR, β2AR.

G protein-coupled receptors (GPCRs) are the most common transmembrane receptors in the human genome and the target of approximately one-third of all approved drugs. GPCRs interact with many transducers like G proteins and β-arrestins. Some GPCRs preferentially activate specific signaling transducers over others, leading to unique signaling profiles – a phenomenon called biased signaling. The chemokine system, a subfamily of GPCRs, serves as an endogenous example of biased signaling where over 50 different chemokines and 20 receptors interact promiscuously. While previous research has shown that chemokines which activate the same receptor can produce different physiologic responses, the mechanisms underlying these findings remain unclear. Using the three endogenous chemokines of the chemokine receptor CXCR3, we investigated two mechanisms underlying biased signaling at GPCRs. First, using mass spectrometry and cell-based assays, we determined that the chemokines induce different amounts and patterns of GPCR phosphorylation which direct CXCR3 engagement with different transducers. Second, we determined that biased signaling is dependent on the specific location of CXCR3, and subcellular signaling regulates inflammation in a mouse model of contact hypersensitivity. Together, we conclude that differential receptor phosphorylation and subcellular signaling are two mechanisms underlying the biased signaling observed at GPCRs.